Bioremediation of sludge using Pseudomonas Aeruginosa

For the bioremediation of sludge, samples were collected from different places near Jalandhar city of the Punjab (India). In order to analyze the sludge, the physiochemical parameters like pH, Moisture and heavy metal are determined. The bacterial strain Pseudomonas aeruginosa was screened for the removal of heavy metal like LEAD and COPPER from the industrial sludge. The effect on pH and moisture was determined. Maximum lead removal was noted to be 0.20 by Pseudomonas aeruginosa species from the sludge sample and copper removal was noted to be 0.30 by P. aeruginosa species. The present study depicts that the bacterial species remove heavy metal from sludge and can be used for the industrial waste management and other environmental maintenance.

Original Research Article https://doi.org/10.20546/ijcmas.2019.804.009

Bioremediation of Sludge using Pseudomonas aeruginosa

Rajveer Kaur 1* , Gurjot Kaur Mavi 2 and Shweta Raghav 3

1

CT Institute of Pharmaceutical Sciences Jalandhar, India

2

Department of Animal Genetics and Breeding, 3 Department of Veterinary Anatomy,

GADVASU Ludhiana, India

*Corresponding author

A B S T R A C T

Introduction

Bioremediation is the process of utilizing

living organisms, microorganisms to degrade

pollutants and contaminants from the

environment and transform them into less

toxic form Bioremediation is based on the

ability of a microorganism to degrade the

hydrocarbons into components that can be

taken up by other micro-organisms as a

nutrient source or can be safely returned to

the environment Degraded organic

components are converted into water,

carbondioxide and other inorganic

compounds Not only microbes but plants too

help in biodegradation of hydrocarbons An

effective bioremediation requires enzymatic

attack by microorganisms to convert pollutants into harmless products Environmental parameters should be optimum

to help the microorganisms to grow and degrade the pollutants at a rapid rate (de la

Cueva et al., 2016) There are limitations to

this technology also, for example, chlorinated hydrocarbons or other high aromatic hydrocarbons are almost resistant to microbial degradation or are degraded at a really slow pace But bioremediation techniques are somewhat economical and can be widely implemented Most of the techniques in bioremediation are aerobic in nature, but anaerobic processes are also being developed

to help degrade pollutants in oxygen deficit

areas (Franchi et al., 2016) There are two

International Journal of Current Microbiology and Applied Sciences

ISSN: 2319-7706 Volume 8 Number 04 (2019)

Journal homepage: http://www.ijcmas.com

For the bioremediation of sludge, samples were collected from different places near Jalandhar city of the Punjab (India) In order to analyze the sludge, the physiochemical parameters like pH, Moisture and heavy metal are determined

The bacterial strain Pseudomonas aeruginosa was screened for the removal of

heavy metal like LEAD and COPPER from the industrial sludge The effect on pH and moisture was determined Maximum lead removal was noted to be 0.20 by

Pseudomonas aeruginosa species from the sludge sample and copper removal was

noted to be 0.30 by P aeruginosa species The present study depicts that the

bacterial species remove heavy metal from sludge and can be used for the industrial waste management and other environmental maintenance

K e y w o r d s

Bioremediation,

Pseudomonas

aeruginosa,

Sludge

Accepted:

04 March 2019

Available Online:

10 April 2019

Article Info

types of bioremediation strategies: In Situ

Bioremediation- This method of

bioremediation is cost effective and causes

less disturbance to the surrounding area of the

contaminated site In situ method is mainly

used for soil contamination due to oil spills

Thus, it is limited by the depth up to which

microorganisms can help degrade pollutants

Mostly upto 30-60 cm of depth in soil have

been reached for the process of

bioremediation Bioventing- This is common

in situ method of bioremediation which

involves supplying air at a low flow rate and

provides much-needed oxygen and nutrients

by wells to stimulate the microorganisms in

the contaminated site Through this method, it

is determined that volatilization of the

contamination is avoided, and they do not

spread It is an effective method for simple

hydrocarbons In situ biodegradation- In this

process oxygen and nutrients are added to the

soil by means of an aqueous solution that

circulates through the contaminated soil, the

solution contains water-containing nutrients,

oxygen and electron acceptors to stimulate the

microorganism This method is used for soil

and groundwater treatment

Ex-situ bioremediation- Biopiles- This is a

combination of landfarming and composting

In this method engineered cells are

constructed in composted piles in a

well-aerated condition Moreover, this technique is

refined from land farming method and

controls the spread of contamination by

volatilization and leaching This technique is

used for treatment of contamination of the

surface of spilled hydrocarbon pollutants

mainly petroleum products Biopiles helps

grow indigenous aerobic and anaerobic

microorganisms Apart from biopiles, Land

farming and composting are two other

methods of Ex-situ bioremediation

Bioreactors- Bioreactors are used for treating

hydrocarbon pollutants in a safe and simple

way It is used for ex situ bioremediation

where slurry reactors or aqueous reactors are used for treating contaminated water or soil The contaminants are kept in a containment vessel and using various apparatus mixing is done at a three-phase system that is, solid, liquid and gas The slurry formed due to this mixing help the biodegradation of the pollutants and also increases the biomass (which contains the microorganisms) The only limitation of this technique is that the pretreatment that has to be done before the contaminated soil or water can be placed in

the bioreactors (Vidali, 2001)

Bioremediation has been proven to be an effective, environmentally friendly and less expensive treatment option for remediation of aquifers contaminated with hydrocarbons (Shen and Wang, 1995; Jardine and Taylor, 1995; Ganguli and Tirupathi, 2002) Biotransformation is the process by which a highly toxic compound is converted to less toxic/no toxic compound using biological process This process can be aerobic / anaerobic / anoxic or combination of these three, based on the microorganisms It has been reported that several microorganisms, under various environmental conditions, can decontaminate the dam sediments very effectively

This process depends on carbon source, pH, temperature, dissolved oxygen, ORP, and presence of other oxyanions and metal cations (Chen and Hao, 1998) Bioremediation is the use of biological treatments, for the clean-up

of hazardous chemicals in the environment

At present, employing the, biochemical abilities of microorganisms is the most popular strategy for the biological treatment

of contaminated soils, sediments and waters (Head, 1998) Now a days, great emphasis is placed on environmental biotechnology and attaining sustainable development: in particular, biological techniques can be applied effectively in the remediation of

sediments contaminated by organic pollutants

from a variety of sources

Bioremediation can be defined as a natural or

managed biological degradation of

environmental pollution The indigenous

microorganisms normally carry out

bioremediation and their activity can be

enhanced by a more suitable supply of

nutrients and/or by enhancing their

population Therefore, this process exploits

such microorganisms and their enzymatic

activities to effectively remove contaminants

from contaminated sites This process is a

cost effective means of cleanup of

hydrocarbon spills from contaminated sites as

it involves simple procedures only and it is an

environmentally friendly technology which

optimizes microbial degradation activity via

control of the pH, nutrient balance, aeration

and mixing Also, bioremediation is a

versatile alternative to physicochemical

treatments and produces non-toxic end

products such as CO2, water and methane

from petroleum hydrocarbons

In situ bioremediation

The most effective means of implementing in

situ bioremediation depends on the hydrology

of the subsurface area, the extent of the

contaminated area and the nature of the

contamination In general, this method is

effective only when the subsurface soils are

highly permeable, the soil horizon to be

treated falls within a depth of 8-10 m and

shallow groundwater is present at 10 m or less

below ground surface

On site (ex site) bioremediation

Here the contaminated soil is excavated and

placed into a lined treatment cell Thus, it is

possible to sample the site in a more thorough

and, therefore, representative manner On site

treatment involves land treatment or land

farming

Microorganism used for bioremediation

Pseudomonas aeruginosa (ATCC NO.2453)

Kingdom Bacteria Phyllum Proteobacterium Class Gamma Proteobacter

Order Pseudomonadace Family Pseudomonadaces Genus Pseudomonas

Spcies P.aeruginosa

bacterial strain)

Effluents released from textile industries contain various organic dyestuffs, chrome and other chemicals during various operations and produce a large quantity of solid and liquid waste containing hexavalent chromium, salts

of zinc, sulfate, copper, lead The treatment of these wastes is essential before discharging them to environment because of the toxicity and carcinogenicity In trace amounts the lead

is considered as essential nutrient but it is more carcinogenic and mutagenic at elevated level It is also toxic to humans and plants Conventional treatment technologies become less effective and more expensive when metal concentration are in form of 10 to 100mg/1 Non conventional technologies are proved to

be effective in removal of metal under this range such as 99.9% of lead was removed in the10gm/1 of lead solution

However the conventional and less effective physico chemical method are being replaced

by more effective biological methods such as biosorption for the removal of hexavalent lead from the aqous solution, biostimulation for the lead (V1), bioreduction for lead contamination in sludge and ground water bu the reducing bacteria which include the use of eco friendly and easy available material such

as cocoa shells, peanut shells that are removed hexavalent lead activity

In the present study Pseudomonas aeruginosa

strain is used for the removal of heavy metals

like lead, copper, cadmium and chromium

This bacteria is found very effectively in

bioremediation of heavy metal because metals

are directly and indirectly involved in the all

aspect of microbial growth metabolism

Bioremediation of heavy metal by bacterial

cell has been recognized as potential

alternative to existing technologies for the

removal of heavy metal from the industrial

waste This study is an attempt to explore

innovative, cost effective and environment

friendly technology for the bioremediation of

lead contamination using microorganisms

Materials and Methods

For the bioremediation of lead sludge sample

were collected from the dumping site and

nearby area of textile industries

Analysis of sludge sample

The physical chemical parameters (Ph, color,

moisture, phosphorus and heavy metal like

lead and copper) were determined Ph is

determined by electronic digital ph meter

Phosphorus was determined by the

colorimeter lead was determined by

colorimeter and Copper was determined by

colorimeter for metal analysis the effluent

sample were digested with HNO3

Revival of lyophlized culture

From pre-scored ampules

Disinfect the sample by wiping with 70%

alcohol

Wrap the scored area (arrow at the narrow

neck below the gold colored band) with the

ethanol dampened tissue to protect your

fingers The tissue should not so wet that

alcohol enters the ampoule

Bend and break the ampoule at the narrow,

pre scored area The alcohol damped tissue provides good cushioning and protection against cuts for this step

Aseptically added 0.2-0.5 ml of sterile water Using a sterile pipette gently aspired the contents several times to mix the suspension thoroughly

Let the suspension to rest for 15-30 minutes Inoculate the suspension onto an appropriate medium and incubate

Results and Discussion

The sample was taken from various drains surrounding Jalandhar and as well as from the dumping site of industries Then they were analyzed for physico-chemical properties such as pH, moisture content, phosphorus and heavy metal like lead and copper) The pH of the sludge was varied according to their origin ranging between 5.6 and 8.9 The ph was determined by using calibrated ph meter The higher value of moisture and solid were observed in sample collected from Hamira and Kala sanghian drains The bacterial cultures exhibited removal even at higher levels of lead and the bacterial growth decreased with increase in the metal concentration Similarly, sludge samples were

analyzed for heavy metals Nine different

bacterial species were screened on the basis of morphological characteristics which grew in 10-50 mg/l of lead concentration After

screening, Pseudomonas aeruginosa was

found capable to remove lead and used for further study It showed consistent growth, both in nutrient broth and nutrient The data was observed for the uptake of metal ions vs contact time for different conc The metal removal efficiency increased with increase in time However, a remarkably increased in percent lead removal was estimated 75.0 ±

2.27% by Pseudomonas species Different

concentration of both was used like 10mg/l,

and 69.70 ± 0.80% removal by Pseudomonas aeruginosa at 40mg/l and 90.88 ± 0.87 % by

Pseudomonas aeruginosa Pseudomonas

aeruginosa removed considerable amount of

lead and showed significant efficiency for

bioremediation From the above results it is

observed that Pseudomonas aeruginosa can

be used for the removal of lead from waste

generated by industries Further study can be

carried out different concentrations and the

strain can be selected for further removal of

lead from effluent and sludge pH of active

sludge effluent was 8.0 and atmospheric

temperature was 25°C, while ambient

temperature was 20°C Several mesophilic

gram-negative and copper resistant bacteria

were also isolated The enrichment media

showed better growth in comparison to direct

culture method for the isolation of copper

resistant bacteria and less time was taken by

the organisms Also, the isolates in primary

enrichment method could grow on 6 mM

concentration of Copper containing medium

Majority of the bacterial isolates were belonging to gram-negative non-fermentive

Pseudomonas (4 isolates) One gram-negative

coccus was also capable to grow on 2mM concentrations of Copper; but on subsequent inoculation, the strain lost its ability to grow

on more than 2ml copper The data obtained

in this study clearly shows that with use of cadmium resistant mutated biomass, bioaccumulation of Copper solution

considerably increased P.aeruginosa one of

the isolate was able to efficiently remove 94.7% in 30 mg/L of copper solution within

60 min cadmium toxicity Bio-chemical tests were performed to characterize microorganisms on basis of morphological and biochemical properties The tests performed in the present study were Gram staining, Citrate utilization test, H2S production, Nitrate reduction test, Indole test, Methyl red test, Voges-proskaeur test

6.6

0.77

0.190.28 0.180.33 0.19 0.24 0.220.19 0

1

2

3

4

5

6

7

8

MAQSUDAN

PH PHOSPHORUS LEAD

COPPER

0.61

0.170.29 0.350.28 0.340.26 0.16 0.19 0

1

2

3

4

5

6

7

CHAHERU

PH PHOSPHORUS LEAD

COPPER

7

0.77

0.26

0.67

0.29

0

1

2

3

4

5

6

7

8

BASTI BAWA

PH PHOSPHORUS LEAD

COPPER

6.5 6.6 6.6 6.5

0.86

0

1

2

3

4

5

6

7

KALA SANGHIA

PH PHOSPHORUS LEAD

COPPER

6.5

0

1

2

3

4

5

6

7

8

9

URBAN ESTATE

PH PHOSPHORU S

LEAD COPPER

0.37 0.32 0.50.26 0.490.36 0.150.37 0

1

2

3

4

5

6

7

8

9

HAMIRA

PH PHOSPHORU S

LEAD

Oleszkiewicz et al., (1993) described the

moisture content by dry method of sludge in

which sludge drying is really a necessity,

through discussing the results of sludge

drying, the process of sludge drying

Shiro Yoshizaki et al., (2000) worked on

Principle and Process of Heavy Metal

Removal from Sewage Sludge The sufficient

removal of heavy metals from sewage sludge

remains to be achieved

Hulsbeek et al., (2002) described a protocol

for improving the quality and controllability

of the simulation studies for activated sludge

processes

Zespół et al., (2004) explained about

economical factors of sludge by using dry

method Sludge utilization options often

indicate sludge drying as the best option

Yang et al., (2005) discus about the potential

adsorbent for phosphorus removal Alum

sludge refers to the byproduct from the

processing of drinking water in Water

Treatment Works

Zheng et al., (2009) described the Sludge

Phosphorus Tests in Phosphorus is an essential element for plant growth and development, as it plays key roles in plant metabolism, structure and energy transformation

Jabbari Nezhad Kermani et al., (2010)

describe that Lead bioremediation by metal-resistant mutated bacteria isolated from active

sludge of industrial effluent in which

Bioremediation of metal pollutants from industrial wastewater using metal resistant bacteria is a very important aspect of environmental biotechnology

Wenyi Deng et al., (2010) reported on

Moisture distribution in sludges based on different testing methods in which Moisture distributions in municipal sewage sludge and dyeing sludge and paper mill sludge were experimentally studied based on four different methods, i.e., drying test, thermogravimetric

Krishnaveni et al., (2013) explained that

bioremediation of steel industrial effluents using sludge microorganisms in which

Bioremediation is treatment processes that

uses naturally occurring microorganisms as

well as plants to breakdown, or degrade

hazardous substances into less toxic or non

toxic substances

Ghazali et al., (2004) investigated the

bioremediation of hydrocarbon in

contaminated soils by mixed cultures of

hydrocarbon-degrading bacteria The strains

were selected based on the criteria that they

were able to display good growth in crude oil,

individual hydrocarbon compounds or both

Their ability to degrade hydrocarbon

contamination in the environment was

investigated using soil samples that were

contaminated with diesel, crude oil 52 or

engine oil

Vezzulli et al., 2004 evaluated the potential of

bioremediation for mobilisation of carbon in

organic-rich sediments Both bioaugmentation

(bio-fixed microorganisms) and

biostimulation (oxygen release compounds

ORC) protocols had been tested and the

response of the bacterial community has been

described to assess the baseline for

bioremediation potential

Mrayyan and Battikhi (2005) described

bioremediation as cost effective,

environmentally friendly treatment for oily

contaminated sites by the use of

microorganisms In their study, laboratory

experiments were conducted to establish the

performance of bacterial isolates in

degradation of organic compounds contained

in oily sludge from the Jordanian Oil Refinery

plant As a result of the laboratory screening,

three natural bacterial consortia capable of

degrading total organic carbons (TOC) were

prepared from isolates enriched from the oil

sludge

Shuchi et al., (2006) tested the ability of three

bacterial strains, Bacillus sp SV9,

Acinetobacters., SV4 and Pseudomonas sp

SV17 from contaminated soil in Ankleshwar, India to degrade the complex mixture of petroleum hydrocarbons (such as alkanes, aromatics, resins and asphaltenes), sediments, heavy metals and water known as oily sludge

Margesin et al., (2005) evaluated soil

biological activities as a monitoring instrument for the decontamination process of

a mineral oil contaminated soil was made using measurements of microbial counts, soil respiration, soil biomass and several enzyme activities

The sample was taken from various drains surrounding Jalandhar and as well as from the dumping site of industries Then they were analyzed for physico-chemical properties such as pH, moisture content, phosphorus and heavy metal like lead and copper) The pH of the sludge was varied according to their origin ranging between 5.6 and 8.9 The ph was determined by using calibrated ph meter The higher value of moisture and solid were observed in sample collected from Hamira and Kala sanghian drains The bacterial cultures exhibited removal even at higher levels of lead and the bacterial growth decreased with increase in the metal concentration Similarly, sludge samples were

analyzed for heavy metals Nine different

bacterial species were screened on the basis of morphological characteristics which grew in 10-50 mg/l of lead concentration After

screening, Pseudomonas aeruginosa was

found capable to remove lead and used for further study It showed consistent growth, both in nutrient broth and nutrient The data was observed for the uptake of metal ions vs contact time for different conc The metal removal efficiency increased with increase in time However, a remarkably increased in percent lead removal was estimated 75.0 ±

2.27% by Pseudomonas species Different

concentration of broth was used like 10mg/l,

and 69.70 ± 0.80% removal by Pseudomonas

aeruginosa at 40mg/l and 90.88 ± 0.87 % by

aeruginosa removed considerable amount of

lead and showed significant efficiency for

bioremediation From the above results it is

observed that Pseudomonas aeruginosa can

be used for the removal of lead from waste

generated by industries Further study can be

carried out different concentrations and the

strain can be selected for further removal of

lead from effluent and sludge pH of active

sludge effluent was 8.0 and atmospheric

temperature was 25°C, while ambient

temperature was 20°C Several mesophilic

gram-negative and copper resistant bacteria

were also isolated The enrichment media

showed better growth in comparison to direct

culture method for the isolation of copper

resistant bacteria and less time was taken by

the organisms Also, the isolates in primary

enrichment method could grow on 6 mM

concentration of Copper containing medium

Majority of the bacterial isolates were

belonging to gram-negative non-fermentive

Pseudomonas (4 isolates) One gram-negative

coccus was also capable to grow on 2mM

concentrations of Copper; but on subsequent

inoculation, the strain lost its ability to grow

on more than 2ml copper The data obtained

in this study clearly shows that with use of

cadmium resistant mutated biomass,

bioaccumulation of Copper solution

considerably increased P.aeruginosa one of

the isolate was able to efficiently remove

94.7% in 30 mg/L of copper solution within

60 min cadmium toxicity Bio-chemical tests

were performed to characterize

microorganisms on basis of morphological

and biochemical properties The tests

performed in the present study were Gram

staining, Citrate utilization test, H2S

production, Nitrate reduction test, Indole test,

Methyl red test, Voges-Proskaeur test

It can be concluded from the present study

“Bioremediation of Sludge using

Pseudomonas aeruginosa has great potential

to remove the heavy metals like lead and copper from the sludge sample The strain of

Pseudomonas aeruginosa can be successfully

used for the removal of lead, copper, cadmium and chromium These bacteria were found very effectively in bioremediation of heavy metal because metals are directly and indirectly involved in the all aspect of microbial growth metabolism Bioremediation

of heavy metal by bacterial cell has been recognized as potential alternative to existing technologies for the removal of heavy metal from the industrial waste This is an attempt

to explore a new innovative, cost effective and environment friendly technology for the bioremediation of sludge containing heavy metals as contaminants by using microorganisms

References

Chen J.M and O.J Hao, (1998), Microbial

chromium (VI) reduction, Crit Rev Environ Sci Technol 28 (3) 219–251

De la Cueva, S C., Rodríguez, C H., Cruz,

N O S., Contreras, J A R., and Miranda, J L (2016) Changes in Bacterial Populations During Bioremediation of Soil Contaminated with Petroleum Hydrocarbons Water, Air, and Soil Pollution, 227(3): 1-12 Franchi, E., Agazzi, G., Rolli, E., Borin, S.,

Marasco, R., Chiaberge, S and Barbafieri, M (2016) Exploiting hydrocarbon‐degrader indigenous bacteria for bioremediation and

multi‐contaminated soil Chemical Engineering and Technology

Ganguli, A and A.K Tripathi (2002),

Bioremediation of toxic chromium from electroplating effluent by